Collagen is a biodegradable protein and exists in a form of fibers in connective tissue of most animals. The primary function of collagen is to maintain the integrity of tissues and to provide tensile strength essential to tissues. Collagen molecule is a biological macromolecule composed of three polypeptide chains that twist around one another. Each polypeptide is composed of about one thousand amino acids, wherein the primary amino acids are glycine, proline and hydroxyproline. At present, more than 21 different types of collagen have been discovered.
For applications, collagen can be manufactured in different forms, such as sponge, gel, tube, sheet, etc. They can be applied as hemostats, wound dressings, drug carriers, scaffolds of artificial organs, fillers to recover tissues, microcarriers and macrocarriers for supporting cell growth, etc. In order to make the above referred collagen matrix exist a porous structure to facilitate cell migration, cell growth or encapsulation and release of drugs, collagen is usually isolated from connective tissue and fabricated into a porous matrix through a lyophilization step. Generally, the matrix is treated with a cross-linking agent or is mixed with a polymer or a polysaccharide prior to or subsequent to the lyophilization step.
The preparation of porous collagen matrix has been disclosed in many prior art patents. For instance, in U.S. Pat. No. 4,193,813, comminuted collagen at pH 3.5 to pH 6.5 is crosslinked with glutaraldehyde followed by freezing at 0 to −20° C. After thawing, the water of the frozen material is eliminated to form a sponge matrix. The pore size of the matrix formed by this process is about 80˜1400 μm.
U.S. Pat. No. 4,412,947 relates to a process that pure insoluble particulate collagen is suspended in a weak aqueous organic acid solution followed by freezing at −60 to −70° C. with a temperature reduction rate of −0.3 to −0.4° C. per minute, and then lyophilized to form a porous collagen sheet. U.S. Pat. No. 4,522,753 relates to a process of mixing collagen and chrondroitin sulfate to form a copolymer material. The material is then cross-linked by glutaraldehyde and lyophilized to form a porous matrix with a pore size of 20˜180 μm. Such matrix can be used as a basic material of synthetic skin grafts.
U.S. Pat. No. 4,970,298 discloses a collagen matrix prepared by dispersing collagen in an acidic solution or by mixing the collagen dispersion with hyaluronic acid and fibronectin. The dispersion is frozen at the different temperatures and then lyophilized to form a porous sponge. The sponge is cross-linked with a carbodiimide or by a dehydrothermal process. The freezing temperature is −30° C. to −50° C. The pore size of the matrix obtained is about 20˜250 μm. The collagen matrix containing hyaluronic acid or fibronectin exhibits a pore size of 100˜150 μm.
U.S. Pat. No. 4,948,540 describes a process that involves freeze-drying the mixture of native collagen and soluble collagen fibers and compressing at a pressure of 15,000˜30,000 p.s.i. The material is then cross-linked by dehydrothermal method to obtain a final product which is a sheet material with high absorptivity.
U.S. Pat. No. 5,116,552 describes a process for preparing a crack-free sponge matrix. An acidic collagen solution is frozen at −40° C. and lyophilized into a sponge. The sponge is then incubated at 105° C. for 24 hours and then cross-linked for 24 hours with glutaraldehyde to form a matrix with a pore size of 50˜120 μm. The matrix is then immersed in 15% alcohol. After second lyophilization at a lower temperature of −80° C. or −135° C., a crack-free sponge matrix is obtained.
U.S. Pat. No. 5,869,080 describes a process for preparing an absorbable implant material. A sponge matrix is formed by adding a proper amount of alcohol to the collagen dispersion in sodium hydroxide, pre-freezing it at a low temperature (about −5° C.), adding is ice particles to the dispersion, cross-linking the dispersion with hexamethylene diisocyanate (HMDI), and followed by lyophilizing the dispersion. The matrix obtained by this process exhibits a pore size of 50˜400 μm.
U.S. Pat. No. 4,320,201 describes a process for preparing a collagen sponge with a velour-like surface and which is insoluble but highly swellable in water. The making process comprises dehairing animal hides, enzymatically degrading the hides to form a collagen-containing mass, digesting the mass in an acid and/or alkali, swelling the mass in acid, comminuting part of the swollen mass to a fiber, forming a paste in water of about 0.5 to 3 weight % of a mixture which on a dry basis, adjusting the pH to about 2 to 3.5, cross-linking the paste in the presence of a cross-linking agent such as aliphatic, aromatic, or hydroaromatic diisocyanates at a temperature below 0° C. to form a swollen sponge, and removing water by freeze drying.
U.S. Pat. No. 5,019,087 describes a process that preparing a hollow conduit comprises of a matrix of type I collagen and laminin-containing material derived from animal collagenous tissue for promoting the in vivo regeneration of a severed nerve wherein laminin-containing material acts the role of cross linker to support collagen strength. The cleaned and washed collagen containing material has to be comminuted, generally by slicing or grinding in this invention.
WO Patent No. 99/13902 describes a process that preparing dural substitutes with collagen dispersions derived from animal tissues for promoting meningeal tissue growth. Suitable forms for the dural substitutes include a sponge, a film, a non-woven matrix, a felt or a combination of at least two of the foregoing forms. The matrix is a planar object having pores ranging in diameter from about 30 μm to 300 μm wherein the cleaned and washed collagen containing material is comminuted generally by slicing or grinding. In this invention, the enhanced strength of products is accomplished by desired rate of bioresorption which is seen as self-cross linking step.
U.S. Pat. No. 5,993,844 describes a detergent-free and enzyme-free method for the removal of non-collagenous components from native mammalian tissues to yield an essentially collagenous matrix that maintains its structure integrity. Although the process uses alkaline/cheating agent solution and acid solution containing salt to remove cells and to relax the collagen fiber, the matrix doesn't have the characteristic of porous structure.
U.S. Pat. No. 5,336,616 discloses a method for processing and preserving an acellular collagen-based tissue matrix for transplantation. The method includes the steps of processing biological tissues with a stabilizing solution to reduce procurement damage, treatment with a processing solution to remove cells, treatment with a cryoprotectant solution followed by freezing, drying, storage and rehydration under conditions that preclude functionally significant damage and reconstitutions with viable cells. The matrix without alkaline and acid treatment also has no pores.
WO Patent No. 98/22153 describes the collagen webs which are formed in situ by dehydration and cross linking of the insoluble collagen at sites of injuries, and to uses thereof in stimulating epithelial migration to assist wound healing. In this invention, the in situ process must be treated by mechanically dissociate to obtain homogenous colloid and further comprising the cross linking process by drying.
These processes for the preparation of the collagen matrix disclosed in the aforementioned patents comprise complicated operation processes which include comminuting starting materials generally by slicing or grinding, extraction, purification, lyophilization, and further comprising cross-linking process. Acidic or alkaline collagens are generally used and cross-linked by a dehydrothermal process or by some chemical cross-linking agents, and lyophilized to obtain porous collagen matrices. The matrix products obtained by any of these processes exhibits poor porous homogeneity. Furthermore, because most of the chemical cross-linking agents are toxic, the application extent of these processes is limited.
To obtain a better porous homogeneity, to prevent possible toxic effects resulted from the chemical cross-linking agents, to save vast energy on collagen extraction, and yet to obtain a more stable porous network, the subject invention proposes an improved process for manufacturing the excellent porous collagen matrix for collagen-related products and applications.